The active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions

HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.

Optimisation of Banana streak virus (BSV) diagnostic assays

Bananas are one of the world�fs most important crops, serving as a staple food and an important source of income for millions of people in the subtropics. Pests and diseases are a major constraint to banana production. To prevent the spread of pests and disease, farmers are encouraged to use disease�] and insect�]free planting material obtained by micropropagation. This option, however, does not always exclude viruses and concern remains on the quality of planting material. Therefore, there is a demand for effective and reliable virus indexing procedures for tissue culture (TC) material. Reliable diagnostic tests are currently available for all of the economically important viruses of bananas with the exception of Banana streak viruses (BSV, Caulimoviridae, Badnavirus). Development of a reliable diagnostic test for BSV is complicated by the significant serological and genetic variation reported for BSV isolates, and the presence of endogenous BSV (eBSV). Current PCR�] and serological�]based diagnostic methods for BSV may not detect all species of BSV, and PCR�]based methods may give false positives because of the presence of eBSV. Rolling circle amplification (RCA) has been reported as a technique to detect BSV which can also discriminate between episomal and endogenous BSV sequences. However, the method is too expensive for large scale screening of samples in developing countries, and little information is available regarding its sensitivity. Therefore the development of reliable PCR�]based assays is still considered the most appropriate option for large scale screening of banana plants for BSV. This MSc project aimed to refine and optimise the protocols for BSV detection, with a particular focus on developing reliable PCR�]based diagnostics Initially, the appropriateness and reliability of PCR and RCA as diagnostic tests for BSV detection were assessed by testing 45 field samples of banana collected from nine districts in the Eastern region of Uganda in February 2010. This research was also aimed at investigating the diversity of BSV in eastern Uganda, identifying the BSV species present and characterising any new BSV species. Out of the 45 samples tested, 38 and 40 samples were considered positive by PCR and RCA, respectively. Six different species of BSV, namely Banana streak IM virus (BSIMV), Banana streak MY virus (BSMYV), Banana streak OL virus (BSOLV), Banana streak UA virus (BSUAV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), were detected by PCR and confirmed by RCA and sequencing. No new species were detected, but this was the first report of BSMYV in Uganda. Although RCA was demonstrated to be suitable for broad�]range detection of BSV, it proved time�]consuming and laborious for identification in field samples. Due to the disadvantages associated with RCA, attempts were made to develop a reliable PCR�]based assay for the specific detection of episomal BSOLV, Banana streak GF virus (BSGFV), BSMYV and BSIMV. For BSOLV and BSGFV, the integrated sequences exist in rearranged, repeated and partially inverted portions at their site of integration. Therefore, for these two viruses, primers sets were designed by mapping previously published sequences of their endogenous counterparts onto published sequences of the episomal genomes. For BSOLV, two primer sets were designed while, for BSGFV, a single primer set was designed. The episomalspecificity of these primer sets was assessed by testing 106 plant samples collected during surveys in Kenya and Uganda, and 33 leaf samples from a wide range of banana cultivars maintained in TC at the Maroochy Research Station of the Department of Employment, Economic Development and Innovation (DEEDI), Queensland. All of these samples had previously been tested for episomal BSV by RCA and for both BSOLV and BSGFV by PCR using published primer sets. The outcome from these analyses was that the newly designed primer sets for BSOLV and BSGFV were able to distinguish between episomal BSV and eBSV in most cultivars with some B�]genome component. In some samples, however, amplification was observed using the putative episomal�]specific primer sets where episomal BSV was not identified using RCA. This may reflect a difference in the sensitivity of PCR compared to RCA, or possibly the presence of an eBSV sequence of different conformation. Since the sequences of the respective eBSV for BSMYV and BSIMV in the M. balbisiana genome are not available, a series of random primer combinations were tested in an attempt to find potential episomal�]specific primer sets for BSMYV and BSIMV. Of an initial 20 primer combinations screened for BSMYV detection on a small number of control samples, 11 primers sets appeared to be episomal�]specific. However, subsequent testing of two of these primer combinations on a larger number of control samples resulted in some inconsistent results which will require further investigation. Testing of the 25 primer combinations for episomal�]specific detection of BSIMV on a number of control samples showed that none were able to discriminate between episomal and endogenous BSIMV. The final component of this research project was the development of an infectious clone of a BSV endemic in Australia, namely BSMYV. This was considered important to enable the generation of large amounts of diseased plant material needed for further research. A terminally redundant fragment (.1.3 �~ BSMYV genome) was cloned and transformed into Agrobacterium tumefaciens strain AGL1, and used to inoculate 12 healthy banana plants of the cultivars Cavendish (Williams) by three different methods. At 12 weeks post�]inoculation, (i) four of the five banana plants inoculated by corm injection showed characteristic BSV symptoms while the remaining plant was wilting/dying, (ii) three of the five banana plants inoculated by needle�]pricking of the stem showed BSV symptoms, one plant was symptomless while the remaining had died and (iii) both banana plants inoculated by leaf infiltration were symptomless. When banana leaf samples were tested for BSMYV by PCR and RCA, BSMYV was confirmed in all banana plants showing symptoms including those were wilting and/or dying. The results from this research have provided several avenues for further research. By completely sequencing all variants of eBSOLV and eBSGFV and fully sequencing the eBSIMV and eBSMYV regions, episomal BSV�]specific primer sets for all eBSVs could potentially be designed that could avoid all integrants of that particular BSV species. Furthermore, the development of an infectious BSV clone will enable large numbers of BSVinfected plants to be generated for the further testing of the sensitivity of RCA compared to other more established assays such as PCR. The development of infectious clones also opens the possibility for virus induced gene silencing studies in banana.

The inherent international tax regime and its constraints on Australia's sovereignty

Neither an international tax, nor an international taxing body exists. Rather, there are domestic taxing rules adopted by jurisdictions which, coupled with double tax treaties, apply to cross-border transactions and international taxation issues. International bodies such as the OECD and UN, which provide guidance on tax issues, often steer and supplement these domestic adoptions but have no binding international taxing powers. These pragmatic realities, together with the specific use of the word ‘regime’ within the tax community, lead many to argue that an international tax regime does not exist. However, an international tax regime should be defined no differently to any other area of international law and when we step outside the confines of tax law to consider the definition of a ‘regime’ within international relations it is possible to demonstrate that such a regime is very real. The first part of this article, by defining an international tax regime in a broader and more traditional context, also outlining both the tax policy and principles which frame that regime, reveals its existence. Once it is accepted that an international tax regime exists, it is possible to consider its adoption by jurisdictions and subsequent constraints it places on them. Using the proposed changes to transfer pricing laws as the impetus for assessing Australia’s adoption of the international tax regime, the constraints on sovereignty are assessed through a taxonomy of the level adoption. This reveals the subsequent constraints which flow from the broad acceptance of an international tax regime through to the specific adoption of technical detail. By undertaking this analysis, the second part of this article demonstrates that Australia has inherently adopted an international tax regime, with a move towards explicit adoption and a clear embedding of its principles within the domestic tax legislation.

How should Australia regulate voluntary euthanasia and assisted suicide?

The purpose of this paper is to provide a basis from which to start an informed and rational dialogue in Australia about voluntary euthanasia (VE) and assisted suicide (AS). It does this by seeking to chart the broad landscape of issues that can be raised as relevant to how this conduct should be regulated by the law. It is not our purpose to persuade. Rather, we have attempted to address the issues as neutrally as possible and to canvass both sides of the argument in an even-handed manner. We hope that this exercise places the reader in a position to consider the question posed by this paper: How should Australia regulate voluntary euthanasia and assisted suicide? In line with the approach taken in the paper, this question does not take sides in the debate. It simply asks how VE and AS should be regulated, acknowledging that both prohibition and legalisation of such conduct involve regulation. We begin by considering the wider legal framework that governs end of life decision-making. Decisions to withhold or withdraw life-sustaining treatment that result in a person’s death can be lawful. This could be because, for example, a competent adult refuses such treatment. Alternatively, stopping or not providing treatment can be lawful when it is no longer in a person’s best interests to receive it. The law also recognises that appropriate palliative care should not attract criminal responsibility. By contrast, VE and AS are unlawful in Australia and could lead to prosecution for crimes such as murder, manslaughter or aiding and abetting suicide. But this is not to say that such conduct does not occur in practice. Indeed, there is a body of evidence that VE and AS occur in Australia, despite them being unlawful. There have been repeated efforts to change the law in this country, mainly by the minor political parties. However, apart from a brief period when VE and AS was lawful in the Northern Territory, these attempts to reform the law have been unsuccessful. The position is different in a small but increasing number of jurisdictions overseas where such conduct is lawful. The most well known is the Netherlands but there are also statutory regimes that regulate VE and/or AS in Belgium and Luxembourg in Europe, and Oregon and Washington in the United States. A feature of these legislative models is that they incorporate review or oversight processes that enable the collection of data about how the law is being used. As a result, there is a significant body of evidence that is available for consideration to assess the operation of the law in these jurisdictions and some of this is considered briefly here. Assisting a suicide, if done for selfless motives, is also legal in Switzerland, and this has resulted in what has been referred to as ‘euthanasia tourism’. This model is also considered. The paper also identifies the major arguments in favour of, and against, legalisation of VE and AS. Arguments often advanced in favour of law reform include respect for autonomy, that public opinion favours reform, and that the current law is incoherent and discriminatory. Key arguments against legalising VE and AS point to the sanctity of life, concerns about the adequacy and effectiveness of safeguards, and a ‘slippery slope’ that will allow euthanasia to occur for minors or for adults where it is not voluntary. We have also attempted to step beyond these well trodden and often rehearsed cases ‘for and against’. To this end, we have identified some ethical values that might span both sides of the debate and perhaps be the subject of wider consensus. We then outline a framework for considering the issue of how Australia should regulate VE and AS. We begin by asking whether such conduct should be criminal acts (as they presently are). If VE and AS should continue to attract criminal responsibility, the next step is to enquire whether the law should punish such conduct more or less than is presently the case, or whether the law should stay the same. If a change is favoured as to how the criminal law punishes VE and AS, options considered include sentencing reform, creating context-specific offences or developing prosecutorial guidelines for how the criminal justice system deals with these issues. If VE and AS should not be criminal acts, then questions arise as to how and when they should be permitted and regulated. Possible elements of any reform model include: ensuring decision-making is competent and voluntary; ascertaining a person’s eligibility to utilise the regime, for example, whether it depends on him or her having a terminal illness or experiencing pain and suffering; and setting out processes for how any decision must be made and evidenced. Options to bring about decriminalisation include challenging the validity of laws that make VE and AS unlawful, recognising a defence to criminal prosecution, or creating a statutory framework to regulate the practice. We conclude the paper where we started: with a call for rational and informed consideration of a difficult and sensitive issue. How should Australia regulate voluntary euthanasia and assisted suicide?

The family participation in child care project

Working with families has long been a fundamental tenet of quality child care services. While there is broad agreement that family participation in child care offers multiple benefits to all concerned, many educators continue to identify this as one of the more challenging aspects of their work. There are a number of perceived barriers to participation, including time constraints, different needs and expectations and the lack of confidence and capacity to support genuine participation. What is interesting, and often overlooked, is that these are shared issues and relate to both educators and parents. Recognising the importance and challenge of family participation in child care, the Brisbane South Professional Support Network PSN), a network facilitated by the Health and Community Services Workforce Council is leading a collaborative research project to build educator knowledge and capacity to promote and support relationship building, meaningful dialogue and genuine partnerships in child care. This article reports on findings from the first phase of this study, identifying parent views and experiences of partnership and articipation in child care services. Findings highlight preferred methods of information sharing and seeking, identify barriers to communication and participation and provide insight into parent expectations of partnerships with educators.

Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status

BACKGROUND: Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines. RESULTS: The majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201. This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1. CONCLUSIONS: Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.

Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media

Background: HIV-1 Pr55gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability. FINDINGS We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4[degree sign]C, --20[degree sign]C and -70[degree sign]C) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at -70[degree sign]C retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. Conclusions Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at -70[degree sign]C for 12 months is most effective in retaining VLP stability.

HIV-1 subtype C Pr55gag virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine

A DNA vaccine expressing human immunodeficiency virus type 1 (HIV-1) southern African subtype C Gag (pTHGag) and a recombinant baculovirus Pr55gag virus-like particle prepared using a subtype C Pr55gag protein (Gag VLP) was tested in a prime-boost inoculation regimen in Chacma baboons. The response of five baboons to Gag peptides in a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay after three pTHGag immunizations ranged from 100 to 515 spot-forming units (s.f.u.) per 106 peripheral blood mononuclear cells (PBMCs), whilst the response of two baboons to the Gag VLP vaccine ranged from 415 to 465 s.f.u. per 106 PBMCs. An increase in the Gag-specific response to a range of 775-3583 s.f.u. per 106 PBMCs was achieved by boosting with Gag VLPs the five baboons that were primed with pTHGag. No improvement in Gag responses was achieved in this prime-boost inoculation regimen by increasing the number of pTHGag inoculations to six. IFN-γ responses were mapped to several peptides, some of which have been reported to be targeted by PBMCs from HIV-1 subtype C-infected individuals. Gag VLPs, given as a single-modality regimen, induced a predominantly CD8+ T-cell IFN-γ response and interleukin-2 was a major cytokine within a mix of predominantly Th1 cytokines produced by a DNA-VLP prime-boost modality. The prime-boost inoculation regimen induced high serum p24 antibody titres in all baboons, which were several fold above that induced by the individual vaccines. Overall, this study demonstrated that these DNA prime/VLP boost vaccine regimens are highly immunogenic in baboons, inducing high-magnitude and broad multifunctional responses, providing support for the development of these products for clinical trials. © 2008 SGM.

Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism

We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of single-stranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely affected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold differently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic effect, which might have broad implications in the evolution of plants and their viruses.

Global genetic diversity and geographical and host-species distribution of beak and feather disease virus isolates

Psittacine beak and feather disease (PBFD) has a broad host range and is widespread in wild and captive psittacine populations in Asia, Africa, the Americas, Europe and Australasia. Beak and feather disease circovirus (BFDV) is the causative agent. BFDV has an ~2 kb single stranded circular DNA genome encoding just two proteins (Rep and CP). In this study we provide support for demarcation of BFDV strains by phylogenetic analysis of 65 complete genomes from databases and 22 new BFDV sequences isolated from infected psittacines in South Africa. We propose 94% genome-wide sequence identity as a strain demarcation threshold, with isolates sharing > 94% identity belonging to the same strain, and strain subtypes sharing> 98% identity. Currently, BFDV diversity falls within 14 strains, with five highly divergent isolates from budgerigars probably representing a new species of circovirus with three strains (budgerigar circovirus; BCV-A, -B and -C). The geographical distribution of BFDV and BCV strains is strongly linked to the international trade in exotic birds; strains with more than one host are generally located in the same geographical area. Lastly, we examined BFDV and BCV sequences for evidence of recombination, and determined that recombination had occurred in most BFDV and BCV strains. We established that there were two globally significant recombination hotspots in the viral genome: the first is along the entire intergenic region and the second is in the C-terminal portion of the CP ORF. The implications of our results for the taxonomy and classification of circoviruses are discussed. © 2011 SGM.

HIV-1 sub-type C chimaeric VLPs boost cellular immune responses in mice

Several approaches have been explored to eradicate HIV; however, a multigene vaccine appears to be the best option, given their proven potential to elicit broad, effective responses in animal models. The Pr55 Gagprotein is an excellent vaccine candidate in its own right, given that it can assemble into large, enveloped, virus-like particles (VLPs) which are highly immunogenic, and can moreover be used as a scaffold for the presentation of other large non-structural HIV antigens. In this study, we evaluated the potential of two novel chimaeric HIV-1 Pr55 Gag-based VLP constructs - C-terminal fusions with reverse transcriptase and a Tat::Nef fusion protein, designated GagRT and GagTN respectively - to enhance a cellular response in mice when used as boost components in two types of heterologous prime-boost vaccine strategies. A vaccine regimen consisting of a DNA prime and chimaeric HIV-1 VLP boosts in mice induced strong, broad cellular immune responses at an optimum dose of 100 ng VLPs. The enhanced cellular responses induced by the DNA prime-VLP boost were two- to three-fold greater than two DNA vaccinations. Moreover, a mixture of GagRT and GagTN VLPs also boosted antigen-specific CD8+ and CD4+ T-cell responses, while VLP vaccinations only induced predominantly robust Gag CD4+ T-cell responses. The results demonstrate the promising potential of these chimaeric VLPs as vaccine candidates against HIV-1. © 2010 Pillay et al; licensee BioMed Central Ltd.

Is it time to revisit the problem young driver?

For decades there have been two young driver concepts: the „young driver problem‟ where the driver cohort represents a key problem for road safety; and the „problem young driver‟ where a sub-sample of drivers represents the greatest road safety problem. Given difficulties associated with identifying and then modifying the behaviour of the latter group, broad countermeasures such as graduated driver licensing (GDL) have generally been relied upon to address the young driver problem. GDL evaluations reveal general road safety benefits for young drivers, yet they continue to be overrepresented in fatality and injury statistics. Therefore it is timely for researchers revisit the problem young driver concept to assess its potential countermeasure implications. Personal characteristics, behaviours and attitudes of 378 Queensland novice drivers aged 17-25 years were explored during their pre-, Learner and Provisional 1 (intermediate) licence as part of a larger longitudinal research project. Self-reported risky driving was measured by the Behaviour of Young Novice Drivers Scale (BYNDS), and five subscale scores were used to cluster the drivers into three groups (high risk n = 49, medium risk n = 163, low risk n = 166). High risk „problem young‟ drivers were characterised by self-reported pre-Licence driving, unsupervised Learner driving, and speeding, driving errors, risky driving exposure, crash involvement, and offence detection during the Provisional period. Medium risk drivers were also characterised by more risky road use behaviours than the low risk group. Interestingly problem young drivers appear to have some insight into their high-risk driving, and they report significantly greater intentions to bend road rules in future driving. The results suggest that in addition to broad countermeasures such as GDL which target the young driver problem, tailored intervention efforts may need to target problem young drivers. Driving behaviours and crash-involvement could be used to identify these drivers as pre-intervention screening measures.

Using the index for inclusion to develop inclusive school communities

This chapter provides an overview of how school communities can work together in processes or review and development to strive towards a more inclusive approach to education. The writers of this chapter have been using a resource called the Index for Inclusion (Booth & Ainscow, 2005, 2011) for a number of years in Australia and in a pilot trail in New Zealand to support education staff in processes of review, with the aim to increase the participation and learning of all students. The resource supports the development of collaborative community processes and defines inclusion as ‘putting values into action’ (Booth & Ainscow, 2011, p.18). The process of review and development for more inclusive and socially just schools supports the development of a school culture, policy and practice where people are valued and treated with respect for their varied knowledge and experiences. In our experience, this resource has been useful to challenge our thinking about education in school communities and in region/districts about inclusive school development. We suggest the Index framework is broad enough to be used in a range of settings and countries. The resource is also useful for pre-service and in-service teacher development to provoke reflection and discussion about inclusion. This chapter provides an overview of the dimensions and framework that inform the Index of Inclusion. We discuss how the Index can be used in school contexts and draw on our own experience to give real examples of how teachers, paraprofessionals, students, principals and parents have experienced the Index when used in their local school communities in Australia and New Zealand. The chapter concludes with some points for discussion to challenge the status quo in schools and to inspire teachers to work towards a more socially just society through making changes at a school level.

Prediction of transcriptional regulatory interactions in bacteria : a comparative genomics approach

Exponential growth of genomic data in the last two decades has made manual analyses impractical for all but trial studies. As genomic analyses have become more sophisticated, and move toward comparisons across large datasets, computational approaches have become essential. One of the most important biological questions is to understand the mechanisms underlying gene regulation. Genetic regulation is commonly investigated and modelled through the use of transcriptional regulatory network (TRN) structures. These model the regulatory interactions between two key components: transcription factors (TFs) and the target genes (TGs) they regulate. Transcriptional regulatory networks have proven to be invaluable scientific tools in Bioinformatics. When used in conjunction with comparative genomics, they have provided substantial insights into the evolution of regulatory interactions. Current approaches to regulatory network inference, however, omit two additional key entities: promoters and transcription factor binding sites (TFBSs). In this study, we attempted to explore the relationships among these regulatory components in bacteria. Our primary goal was to identify relationships that can assist in reducing the high false positive rates associated with transcription factor binding site predictions and thereupon enhance the reliability of the inferred transcription regulatory networks. In our preliminary exploration of relationships between the key regulatory components in Escherichia coli transcription, we discovered a number of potentially useful features. The combination of location score and sequence dissimilarity scores increased de novo binding site prediction accuracy by 13.6%. Another important observation made was with regards to the relationship between transcription factors grouped by their regulatory role and corresponding promoter strength. Our study of E.coli ��70 promoters, found support at the 0.1 significance level for our hypothesis | that weak promoters are preferentially associated with activator binding sites to enhance gene expression, whilst strong promoters have more repressor binding sites to repress or inhibit gene transcription. Although the observations were specific to �70, they nevertheless strongly encourage additional investigations when more experimentally confirmed data are available. In our preliminary exploration of relationships between the key regulatory components in E.coli transcription, we discovered a number of potentially useful features { some of which proved successful in reducing the number of false positives when applied to re-evaluate binding site predictions. Of chief interest was the relationship observed between promoter strength and TFs with respect to their regulatory role. Based on the common assumption, where promoter homology positively correlates with transcription rate, we hypothesised that weak promoters would have more transcription factors that enhance gene expression, whilst strong promoters would have more repressor binding sites. The t-tests assessed for E.coli �70 promoters returned a p-value of 0.072, which at 0.1 significance level suggested support for our (alternative) hypothesis; albeit this trend may only be present for promoters where corresponding TFBSs are either all repressors or all activators. Nevertheless, such suggestive results strongly encourage additional investigations when more experimentally confirmed data will become available. Much of the remainder of the thesis concerns a machine learning study of binding site prediction, using the SVM and kernel methods, principally the spectrum kernel. Spectrum kernels have been successfully applied in previous studies of protein classification [91, 92], as well as the related problem of promoter predictions [59], and we have here successfully applied the technique to refining TFBS predictions. The advantages provided by the SVM classifier were best seen in `moderately'-conserved transcription factor binding sites as represented by our E.coli CRP case study. Inclusion of additional position feature attributes further increased accuracy by 9.1% but more notable was the considerable decrease in false positive rate from 0.8 to 0.5 while retaining 0.9 sensitivity. Improved prediction of transcription factor binding sites is in turn extremely valuable in improving inference of regulatory relationships, a problem notoriously prone to false positive predictions. Here, the number of false regulatory interactions inferred using the conventional two-component model was substantially reduced when we integrated de novo transcription factor binding site predictions as an additional criterion for acceptance in a case study of inference in the Fur regulon. This initial work was extended to a comparative study of the iron regulatory system across 20 Yersinia strains. This work revealed interesting, strain-specific difierences, especially between pathogenic and non-pathogenic strains. Such difierences were made clear through interactive visualisations using the TRNDifi software developed as part of this work, and would have remained undetected using conventional methods. This approach led to the nomination of the Yfe iron-uptake system as a candidate for further wet-lab experimentation due to its potential active functionality in non-pathogens and its known participation in full virulence of the bubonic plague strain. Building on this work, we introduced novel structures we have labelled as `regulatory trees', inspired by the phylogenetic tree concept. Instead of using gene or protein sequence similarity, the regulatory trees were constructed based on the number of similar regulatory interactions. While the common phylogentic trees convey information regarding changes in gene repertoire, which we might regard being analogous to `hardware', the regulatory tree informs us of the changes in regulatory circuitry, in some respects analogous to `software'. In this context, we explored the `pan-regulatory network' for the Fur system, the entire set of regulatory interactions found for the Fur transcription factor across a group of genomes. In the pan-regulatory network, emphasis is placed on how the regulatory network for each target genome is inferred from multiple sources instead of a single source, as is the common approach. The benefit of using multiple reference networks, is a more comprehensive survey of the relationships, and increased confidence in the regulatory interactions predicted. In the present study, we distinguish between relationships found across the full set of genomes as the `core-regulatory-set', and interactions found only in a subset of genomes explored as the `sub-regulatory-set'. We found nine Fur target gene clusters present across the four genomes studied, this core set potentially identifying basic regulatory processes essential for survival. Species level difierences are seen at the sub-regulatory-set level; for example the known virulence factors, YbtA and PchR were found in Y.pestis and P.aerguinosa respectively, but were not present in both E.coli and B.subtilis. Such factors and the iron-uptake systems they regulate, are ideal candidates for wet-lab investigation to determine whether or not they are pathogenic specific. In this study, we employed a broad range of approaches to address our goals and assessed these methods using the Fur regulon as our initial case study. We identified a set of promising feature attributes; demonstrated their success in increasing transcription factor binding site prediction specificity while retaining sensitivity, and showed the importance of binding site predictions in enhancing the reliability of regulatory interaction inferences. Most importantly, these outcomes led to the introduction of a range of visualisations and techniques, which are applicable across the entire bacterial spectrum and can be utilised in studies beyond the understanding of transcriptional regulatory networks.

The challenges in developing a strategic approach to managing diversity

Diversity management is recognised as a major challenge for organizations throughout the world. There is broad acceptance that when it comes to all aspects of workforce management major differences exist among individuals in terms of age, gender, national origin, physical capability, sexuality, religion and others. This chapter discusses the concept, meaning and application of managing that difference or ‘diversity’ through programs known as diversity management. It identifies and discusses the different contextual and theoretical approaches that frame diversity programs found in organizations today. A number of programs within different country contexts are examined. The discussion examines the challenges of diversity management its programs and its outcomes with a view to understanding the lessons learned and recommending future directions.